Current imaging modalities allow detection of tumors composed of approximately 107 cells or in the range of 1 cubic millimeter. Any increase in imaging sensitivity provides valuable advances in tumor detection and also treatment outcomes; however, a major increase in detection sensitivity would provide a radical change in how we might employ imaging in clinical practice. Ultrasound (US) will likely play a significant and expanding role in oncological imaging in the future because it is safe, low-cost, and readily portable. However, due to fundamental resolution limitations, clinical US can only detect tumor masses on the order of a few millimeters, or larger. In order to improve this detection sensitivity a paradigm shift in the US approach for imaging tumors is needed. This project proposes such a shift. It is well known that tumors dramatically distort microvasculature throughout the angiogenic process. Data show that substantial changes in microvasculature structure occur after the arrival of only 10s to 100s of tumor cells, that these changes extend to vessels that are relatively large (hundreds of microns in diameter), and that microvascular changes extend well beyond tumor margins, even soon after the onset of disease. These unique microvascular cancer signatures provide us with a means to overcome traditional resolution limitations which otherwise impair micro-tumor detection. Thus, our innovative response to improving imaging sensitivity to micro-cancers is to detect these microvascular changes, rather than the solid tumor itself. Prior groups have illustrated the potential for this concept using optical microscopy however optical microscopy is inherently non-clinically translatable for this application, and hence we will utilize a novel ultrasound approach. Although previously, ultrasound has not provided utility in assessing changes in microvascular structure, our group has recently implemented a new US imaging technique called Acoustic Angiography which provides supreme signal-to-noise and high resolution for imaging microvessel structure. This new imaging technique thus enables microvessel segmentation and tortuosity quantification. Our first hypothesis is that we can optimize this imaging approach for adequate spatial resolution and depth of penetration for clinical implementation. Our second hypothesis is that we can use acoustic angiography to detect tumor-induced microvascular changes when tumors are at least two to three orders of magnitude smaller than current detection limits. Our third hypothesis is that we can develop current segmentation and analysis algorithms which will characterize microvessel morphology and provide a specific and sensitive classification approach for detecting tissue that is at risk for hosting micro-tumors. Encouraging preliminary data have already illustrated our ability use acoustic angiography to discriminate small tumors and healthy tissue based on an analysis of microvessel morphology alone, and these further studies will enable a comprehensive development of this promising new technology. Our approach will involve in-vitro studies as well as preclinical in-vivo studies using clinically-relevant geneticaly engineered models of breast cancer. PUBLIC HEALTH RELEVANCE: This application is a response to the NCI Provocative Question #13: Can tumors be detected when they are two to three orders of magnitude smaller than those currently detected with in vivo imaging modalities? We propose to evaluate a novel ultrasound imaging and analysis protocol to detect the cancer-induced microvascular changes which evolve as micro tumors grow from currently undetectable sizes (<1,000 cells) to palpable masses (~10^7 cells). The proposed technology represents a promising route to early cancer detection, and will also have substantial significance in differentiation of benign and malignant lesions as well as tumor response to therapy in personalized medicine.